Our previous studies have found that following a high level of noise exposure, outer hair cells (OHCs) die by both apoptosis and necrosis. The prevalence of apoptosis or necrosis is associated with the noise level and post-exposure progression of the cochlear lesion; however, the cellular mechanisms responsible for determining the pathways of OHC death following noise exposure are still not known. This application focuses on the general hypothesis that the propensity of OHCs to die by apoptosis or necrosis is regulated by the energy level and/or oxidative status of dying OHCs. To test this hypothesis, chinchillas will be exposed to an octave band noise centered at 4 kHz at 110 dB SPL for 1 hour. In the first part of the experiment, the activity of succinate dehydrogenase (SDH), an important mitochondrial enzyme that participates in ATP synthesis in the electron transport chain, will be examined in apoptotic and necrotic OHCs after the noise exposure. In the second part of the study, the synthesis of ATP by OHCs will be blocked by intracochlear application of 3-nitropropionic acid (3-NP), an irreversible inhibitor of SDH. The effect of intracellular ATP depletion on generation of apoptosis and necrosis following the noise exposure will be examined. In the last part of the study, the effect of the alteration of the cochlear antioxidant capacity on the death pathways will be examined. The cochlear antioxidant capacity will be manipulated by changing the level of cochlear glutathione (GSH) either with L-buthionine-[S, R]-sulfoximine, a GSH synthesis inhibitor, or with glutathione monoethyl ester, an analog of GSH. These data will help to elucidate the role of the cochlear energy level and the antioxidant level in determining the prevalence of either apoptosis or necrosis after noise exposure. Our long-term goal is to explore effective therapeutic strategies to reduce noise-induced hearing loss. Since necrotic OHCs potentially create a significantly greater level of toxic stress on surviving OHCs through release of intracellular contents, preventing the conversion of apoptosis to necrosis may ameliorate the overall cochlear damage. The knowledge of the biological mechanisms responsible for modulation of the cell death pathways will provide the basis for eventually developing a rational protective strategy.